Secretion of correctly processed human growth hormone in E. coli and Pseudomonas

ABSTRACT

The production of mature hGH In E. coli and Pseudomonas strains transformed by a plasmid which encodes pre hGH (comprising the signal polypeptide and the hormone itself) is described. These prokaryotes process the pre-hGH to cleave the signal sequence and, thereby, produce mature hGH.

FIELD OF INVENTION

This invention relates to the production of human growth hormone (hGH)with its signal peptide (pre-human growth hormone) in E. coli andPseudomonas and processing of the unprocessed (pre) protein by thebacterial host to cleave the signal sequence from the hGH portion of theprotein to produce mature hGH.

BACKGROUND

Human growth hormone (hGH) is secreted in the human pituitary. In itsmature form it consists of 191 amino acids, has a molecular weight ofabout 21,500, and thus is more than three times as large as insulin.Until the advent of recombinant DNA technology, hGH could be obtainedonly by laborious extraction from a limited source--the pituitary glandsof human cadavers. The consequent scarcity of the substance has limitedits application to treatment of hypopituitary dwarfism even though ithas been proposed to be effective in the treatment of burns, woundhealing, dystrophy, bone knitting, diffuse gastric bleeding andpseudarthrosis. In fact, available estimates are that the amount of hGHavailable from tissue is adequate only to serve about 50 percent of thevictims of hypopituitary dwarfism. Thus, no hGH is available for otherapplications.

Recently, it has been shown that hGH can be produced in a recombinanthost cell, specifically E. coli in quantities which would be adequate totreat hypopituitary dwarfism and the other conditions for which it iseffective. See, for example, U.S. Pat. No. 4,342,832. While this advancein the art promises relief to those who suffer the afflictions for whichit offers hope of amelioration, for reasons which are set forth below,the hGH obtained using the process of U.S. Pat. No. 4,342,832 containsat least a substantial amount of hGH to which the amino acid methioninenot found in native hGH is appended at the N-terminal end of theprotein. While there is no evidence that this slightly different hGHwill, in sensitive individuals, cause any important undesirable sidereactions it is, nevertheless, structurally distinct from "mature" hGH.Hormones which differ slightly from those produced by the human body,such as various insulins obtained as tissue extracts of cattle and otheranimals, have been successfully used to treat human disease for manyyears. Nevertheless, the advent of recombinant DNA techniques have madeit possible to obtain insulin of precisely the same amino acid sequenceas that produced by the body. This has been hailed not only as a greatscientific advance but a medical one as well since the availability of aprocess for making human insulin promises to reduce the risk of adverseside reactions attendant with ingestion of animal insulin to those whosuffer diabetes. Therefore, notwithstanding the availability of hGH inan active form which differs only slightly from that occurringnaturally, there remains a need to obtain hGH conveniently which, in itsamino acid content, consists solely of the 191 amino acid sequence ofthe hGH produced by the pituitary. Further, the herein inventiondiscloses the production of met-less hGH in commercially practicableamounts.

The use of recombinant DNA technology to obtain vectors for expressingheterologous DNA in a transformed microbial host is now a wellestablished science. The first successes in this field were achievedusing strains of the gram-negative bacterium E. coli such as E. coliK-12, strain 294.

The use of E. coli as a microbial host for obtaining complexheterologous polypeptides has its limitations however. Relatively smallpolypeptides must be obtained as a fusion protein in which the targetpolypeptide is expressed as part of a larger polypeptide in order toprotect the small protein from degradation by the host cell. For mostpurposes, the small protein produced as a fusion protein must be cleavedin some way from the larger molecule to obtain a useful product.

Large foreign proteins are not degraded by the cell and can be produceddirectly if the gene for their direct expression, including theappropriately placed start codon, is linked to a suitable promoter gene,such as the well known lac promoter. The signal to begin translation ofthe mRNA coding sequence is the AUG generated from the ATG gene codonwhich also codes for the amino acid methionine (Met). Becauseprokaryotes sometimes do not remove the N-terminal Met from theresulting protein, expression of heterologous DNA under control of abacterial promoter and in a bacterial host sometimes results in aprotein whose first amino acid is methionine. Results to date, forexample, with production of hGH in E. coli, have shown that the hostcell has only a limited ability to cleave methionine intracellularly andthere is no convenient way to do so extracellularly. Accordingly, asnoted above, microbially expressed hGH by the process of U.S. Pat. No.4,342,832 leads to a product in which at least a substantial portion ofthe hGH has the appended methionine group which, in some circumstances,may cause the protein to be recognized as a foreign protein when used intherapeutic applications.

Many naturally occurring proteins are initially expressed in theirnormal environment with an additional peptide sequence which permits theprotein to pass through a cellular membrane of the cell in which it ismanufactured. The additional peptide, which is cleaved in this process,is referred to as a "signal" peptide. If a heterologous gene whichincluded the gene for a signal sequence were placed under control of abacterial promoter and the bacterium would cleave the signal sequenceintracellularly, the mature protein without an appended methioninemoiety could be obtained. However, unless cleaved by the host, thesignal sequence actually complicates isolation of the mature proteinsince extracellular cleavage is not easily accomplished.

Efforts to produce "immature" protein, i.e., the protein of interestcoupled to a signal sequence, in E. coli have suggested thatgram-negative bacteria such as E. coli do not effectively process thisprotein to cleave the signal sequence, however. A small proteinpreproinsulin, has been shown to be partly processed to remove thesignal peptide in E. coli. However, no success at all has been obtainedwith large molecules such as fibroblast and leukocyte interferons. Inthe case of fibroblast interferon, no biologically active material wasproduced (Taniguchi, T. et al., Proc. Natl. Acad. Sci. USA 77, 5230-5233(1980)). In the case of the leukocyte interferons, biologically activematerial was produced but was neither transported nor properlyprocessed.

SUMMARY OF THE INVENTION

We have found, unexpectedly, that the gene for pre-hGH, i.e., a genecoding for the 191 amino acids of the mature protein and the 26 aminoacids of its signal peptide, is expressed to give pre-hGH in gramnegative bacteria which is then processed in the cell to cleave thesignal peptide from the mature protein. (Pre-hGH or pre-protein refersto the desired protein containing a signal sequence which, in its nativeenvironment effects secretion of the desired protein.) As a result, hGHis obtained in its mature form, and in an environment in which it isfree of other proteins associated with its native environment. Thus,using the process of the invention it can be obtained free of proteinsof human origin, in commercially useful amounts, and without thesuperfluous methionine in addition to the amino acid sequence of thenaturally occurring protein.

The present invention also provides replicable vectors for theexpression of the gene for the immature protein which can be used inboth E. coli and Pseudomonas and other prokaryotic bacterial species.The invention further provides prokaryotic hosts transformed by suchvectors.

An object of the present invention, therefore, is an improved processfor obtaining hGH by recombinant DNA technology.

Yet another object is to obtain hGH which is free of appended aminoacids not found in the natural form.

The achievement of these and other objects will be apparent from thefollowing discussion of presently preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the amino acid sequence and mRNA sequence of the signalpolypeptide associated with native hGH and FIG. 1B shows theconstruction of the hGH expression plasmid pRPH2.

DETAILED DESCRIPTION OF THE INVENTION

The general approach to the invention is the preparation of anexpression vector or cloning vehicle which is replicable in the hostprokaryote and which contains a DNA sequence which codes for expressionof immature hGH operably connected to a DNA which effects expression. Asused herein, "prokaryote" refers to cells which do not contain a nucleusand whose chromosomal material is thus not separated from the cytoplasm.Prokaryotes include, for example, bacteria but do not include suchnucleated microorganisms as yeast.

Specifically, plasmids were constructed as expression vectors whichcould be used to transform both E. coli and Pseudomonas strains in orderto demonstrate the ability of bacterial hosts to effect expression ofpre-hGH and process it to cleave the signal sequence.

It has been shown previously that the E. coli plasmid pBR322, which isthe basic plasmid modified for expression of heterologous DNA in E.coli, can be maintained stably in Pseudomonas (Ps.) aerugenosa (a.) ifcloned in the broad host range, sulfonamide resistant (Su^(R)),streptomycin resistant (Sm^(R)) plasmid RSF1010, which is also an E.coli plasmid. See Wood et al., J. Bacteriol., 14, 1448 (1981) andSakagouchi, Current Topics in Microbiology and Immunology, 96, 31(1982). Therefore, to obtain plasmids which would code for hGH and whichcould be used to transform both E. coli and Ps. strains, we determinedto prepare hybrid plasmids of pBR322 and pRSF1010 which contained genesfor the expression of that protein.

The construction of a plasmid to code for the expression of immaturehGH, i.e., for the 191 amino acids of hGH linked to the 26 amino acidsof its signal sequence, is shown in FIG. 1B. The construction uses apBR322 derivative, plasmid phGH207-1 described in de Boer, H. et al.Promoters: Structure and Function, eds. Rodriguez, R. and Chamberlin, M.J. (Praeger, New York), 462-481 (1982). The phGH207-1, designated 1 inFIG. 1B, was partially digested with EcoRI and the largest fragmentpurified by electrophoresis on polyacrylamide gel. This fragment wassubjected to second strand synthesis with DNA polymerase Klenow fragmentand ligated with T4 DNA ligase to form a plasmid, phGH207-1*, with aunique EcoRI site at the junction of the trp promoter-ribosome bindingsite and the hGH structural gene. The phGH207-1* was partially cleavedwith PstI and the largest fragment purified by electrophoresis on apolyacrylamide gel. This fragment was subjected to second strandsynthesis with DNA polymerase Klenow fragment and ligated with T4 DNAligase to form plasmid phGH207-1*-APS, designated 2 with a unique PstIsite within the hGH structural gene. The phGH207-1*-APS was digested tocompletion with EcoRI and PstI and the largest fragment purified by gelelectrophoresis using a polyacrylamide gel. This fragment contains thetrp promoter and the 5' end of the gene of mature hGH.

A second plasmid, phGHcDNA designated 4 in FIG. 1B, was prepared asdescribed by Martial et al., Science 205, 602-606 (1979) and treatedwith HpaII to excise a 462 base pair (bp) fragment which codes for mostof the signal peptide DNA sequence of hGH and the amino terminal portionof mature hGH. This fragment was purified by gel electrophoresis usingpolyacrylamide gel. This fragment was digested with PstI to excise a 192bp fragment which codes for most of the signal peptide DNA sequence ofhGH and the amino-terminal portion of mature hGH. This fragment waspurified by gel electrophoresis using polyacrylamide gel.

Referring now to FIG. 1A, there is shown the amino acid and mRNAsequences for the signal polypeptide of immature hGH with codons AUG forf-Met, the signal for initiation of translation in bacteria, whichreveals that the HpaII site is near the 5' end. To complete the gene forthe signal sequence and to provide EcoRI and HpaII sites, the twosynthetic oligonucleotides 2 in FIG. 1B were synthesized by the improvedtriester method of Crea, Proc. Nat'l. Acad. Sci. USA, 75, 5765 (1978).Aliquots of 5 ug of each of the two synthetic oligonucleotides werephosphorylated using T4 polynucleotide kinase and [γ-³² P]ATP (NEN) asdescribed by Goeddel et al, Proc. Nat'l. Acad. Sci. USA, 76, 106 (1979).The two molecules were then annealed by mixing these reaction products,heating for 5 min. at 90° C. and then cooling to 22° C.

The fragments phGH207-1 and phGHcDNA and the synthetic oligonucleotideswere, in a three way ligation using T4 ligase, joined to obtain plasmidpPrehGH207-1, designated 5 in FIG. 1B, which was cloned in E. coli K-12,strain 294 (E. coli 294), which has been deposited with the AmericanType Culture Collection, ATCC Accession No. 31446, on Oct. 28, 1976. Theplasmid was isolated from a colony which was Tc^(R). The nucloeotidesequence of the region comprising the trp promoter and the codons forthe hGH signal polypeptide and the amino terminal amino acids wasconfirmed by the dideoxy chain termination method. See Messing, J.,Crea, R., and Seeburg, P. H., Nucleic Acids Res. 9, 309-321 (1981).

The pPrehGH207-1 and phGH207-1 were digested with XbaI and BamHI. Thesmaller fragment of pPrehGH207-1 was purified by gel electrophoresis andcontains the entire pre hGH gene. The larger fragment of phGH-207-1 waspurified by gel electrophoresis and contains the trp promoter. These twofragments were mixed and treated with T4 DNA ligase to give plasmid 6,designated pPrehGH207-2, which contains the trp promoter and the pre hGHgene. The pPrehGH207-2 and pRSF1010 designated 7 were treated with EcoRIand joined with T4 ligase to give plasmid 8, designated pRPH-2, whichwas used to transform E. coli 294. The pRPH-2 was obtained from a colonyexhibiting Tc^(R) and Sm^(R).

The expression vector pRPH-2 was used to transform Ps.a. strain PA02003,Chandler et al, Mutat. Res., 23, 15 (1974). Cells transformed withpRPH-2 were grown overnight in Luria broth (LB) with 50 μg/mltetracycline at 37° C. to logarithmic phase. Cell pellets wereresuspended in 30 mM Tris, pH 8.0, 20Δsodium dodecyl sulfate (SDS) andsonicated. The cell extracts were serially diluted for analysis byradioimmunoassay using a Phaedabas hGH PRIST kit (Pharmacia).Substantial levels of hGH were found (4×10² ng/ml/A₅₅₀). Cell extractswere electrophoresed in 12.5 percent SDS on polyacrylamide gels. hGH wasvisualized by an immunoblotting technique using anti-hGH rabbitanti-serum (supplied by Kabi) and ¹²⁵ I-labeled protein a. The cellextracts were shown by autoradiography to contain one major componentreactive with anti-hGH which has the same electrophoretic mobility asauthentic pituitary hGH. A minor band of somewhat lesser mobility wasalso detected and is presumably unprocessed pre hGH.

The major reactive component of the cell extracts was purified tohomogenity by immunoaffinity chromatography and high performance liquidchromatography, and the amino acid sequence of the amino-terminus wasdetermined by the Edman degradation method. See Edman et al, European J.Biochem., 1, 80 (1967). It was found to be homogeneous and to begin withthe sequence Phe-Pro-Thr-Ala in perfect correspondence to the sequenceof pituitary hGH.

Expression of pre hGH accompanied by proteolytic cleavage to obtain themature hGH using pRPH-2 transformants of E. coli and Ps. putida has alsobeen accomplished indicating that the ability of gram negative bacteriato successfully process the pre hGH, although unexpected in view of theprior failure of E. coli to process interferons possessing their ownsignal peptides, is a general phenomenon.

In view of the foregoing those skilled in the art can appreciate thatmodifications of the preferred embodiments can be made without departurefrom the spirit of the invention. For example, it is not necessary touse a plasmid of pRSF1010 if the recombinant host to be employed is E.coli. Thus, successful expression in E. coli can be achieved using, forexample, pPrehGH207-1 or pPrehGH207-2 of FIGS. 1A and 1B. Accordingly,the present invention should be limited only by the lawful scope of theappended claims.

We claim:
 1. A method comprising (a) transforming a procaryotic cellwith an expression plasmid encoding a pre protein for human growthhormone containing a signal sequence, culturing the transformed cell toprovide a prokaryotic recombinant host cell culture comprising maturehuman growth hormone, which growth hormone is free of mature humangrowth hormone having an extraneous N-terminal methionine, and (b)isolating the mature human growth hormone from a cell extract of thehost cell culture of step (a).
 2. The method of claim 1 wherein thesignal sequence comprises the sequencefmet-ala-thr-gly-ser-arg-thr-ser-leu-leu-leu-ala-phe-gly-leu-leu-cys-leu-pro-trp-leu-gln-glu-gly-ser-ala.